Novel cell therapies promise to be highly potent solutions for unmet medical needs, including liver failure, diabetes, and other diseases. To meet the anticipated demand, large numbers of primary cells will often be needed. In particular, mesenchymal stromal cells (MSCs) are a promising cell type for therapeutic applications. Increased cell yield per donor would significantly impact the ability of cell therapy companies including Hepatx to scale manufacturing and provide a commercially viable therapeutic product. However, production of commercial quantities of MSCs while maintaining therapeutic potency is challenging. Human MSCs lack endogenous telomerase activity and the resultant telomere shortening causes them to senesce and lose function. Introduction of telomerase reverse transcriptase (TERT) to MSCs preserves the growth, differentiation, and immunomodulatory effects and can make MSCs amenable to scaled up manufacturing processes. We will employ a recently developed method of rapidly and transiently extending telomeres by delivery of TERT that has several advantages for telomere extension: it is transient, rapid, non-immortalizing, hypoimmunogenic, and safe. Here we propose to develop scalable, GMP compatible methods for transient introduction of TERT mRNA that will extend telomeres to increase the functional lifespan and therapeutic utility of MSCs. Hepatx in collaboration with Rejuvenation Technologies, Inc. will develop the processes necessary to extend telomeres in cultured MSCs at sufficient scale to enable development of larger tiered cell banks, then validate that the expanded MSCs maintain their native multilineage differentiation potential and immunosuppressive activity. Aim 1: Define optimal conditions for extension of telomeres in a bioreactor setting. We will optimize procedures for transfection of RTI?s transient telomere extension formulation into MSCs with high efficiency and viability. Once optimal conditions for transfection have been determined, telomerase activity will be measured. Compatibility with liter-scale bioreactor conditions will be verified. Aim 2: Characterize biological performance of ASC treated with TERT nanoparticles Using the optimized transfection method from Aim 1 we will deliver TERT mRNA to MSCs and measure their proliferative capacity and verify that the treated MSC retain capacity to differentiate into hepatocytes, adipocytes, chondrocytes, osteoblasts. Angiogenic cytokine production will be assayed and immunosuppressive activity will be measured. Pilot testing of novel tiered cell banking processes will be performed. This approach can offer greater expansion of ASC for not only Hepatx?s MSC-derived product, but also for other cell-based therapeutic products. If successful, these studies will demonstrate that MSC production can be scaled without loss of function. A phase II SBIR will develop GMP-compliant methods and extend the results to other cell types.